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A nearly perfect restoration of fast and slow myofibers is accomplished at 30 to 45 dpci. Higher magnifications of cross sections displaying the injured side of mylz2:EGFP and smyhc1:LY-Tomato double transgenic fish. Sections were immunostained for Tnnt2 (slow muscles) and for Tomato. Scale bar indicates 100 μm. a At 10 dpci, all three markers are activated in the wounded area, suggesting advancing regeneration. Most mylz2:EGFP positive fibers (fast myofibers) are negative for LY-Tomato and Tnnt2 (green arrows). The plasma membrane of superficial muscles is immunoreactive for the Tomato antibody (smyhc1:LY-Tomato; red arrows). Some of these cells also express Tnnt2. A few LY-Tomato positive cells are also positive for mylz2:EGFP (yellow arrows). b At 30 dpci, the wounded tissue is filled with muscle fibers. A few mylz2:EGFP single positive myofibers spread into the superficial muscle (green arrows). Some mylz2:EGFP positive myofibers also express smyhc1:LY-Tomato (yellow arrows). Most Tnnt2-positive cells are also Tomato positive cells (purple arrows). c At 45 dpci, nearly no overlap between mylz2:EGFP and smyhc1:LY-Tomato expression suggests terminal differentiation of fast and slow muscle identities. Most of mylz2:EGFP positive fibers retract from the superficial layers, and only a very few individual cells intermingle in the slow muscle compartment (green arrow). Tnnt2 positive cells (blue arrows) form a wedge-like compartment, whereas Tnnt2/LY-Tomato accumulate at the superficial layers, thus, both of which return to the original pattern. Only individual cells that are Tnnt2 or/and LY-Tomato positive remain mislocalized in the fast muscle compartment. Thus, the normal strict separation between fast and slow muscle fibers is still not perfectly re-established. d−g Quantification of fluorescent areas within the wounded area (left side) compared to its respective area of the control side (right side), as indicated on the graphs. White points, uninjured side; black points, cryoinjured side. N = 3 fish, each representing an average of 3 nonadjacent sections. Error bar, SEM. Kruskal-Wallis test with Dunn’s post hoc analysis.
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